P-glycoprotein (P-gp or ABCB1) is a membrane-bound active transporter belonging to the ABC protein superfamily. It is responsible for xenobioIc efflux and also contributes to multidrug resistance in diverse diseases including cancer and epilepsy. P-gp has been increasingly recognised as a potential target for future therapeutics. Although the protein has been studied for decades, understanding of the P-gp transport mechanism is still incomplete. Two P-gp orthologues, mouse (m) and human (h), were therefore expressed in yeasts and purified in the presence of the detergent, n-Dodecyl-β-D- Maltoside (DDM). Purified proteins were examined for aggregation and monodispersity via dynamic light scattering (DLS) and their thermal stability was determined by an assay using a thiol-specific dye (CPM). ATPase activity, measured in a detergent environment, showed that the proteins were active with a basal activity of 60 ± 4 and 35 ± 3 nmol/min/mg for mP-gp and hP-gp, respectively. Crystallisation trials were conducted in the presence of nucleotide. In meso crystallisation using commercial monoolein pre- dispensed plates yielded hexagonal crystal-like objects however they failed to diffract X- rays. P-gp samples were also subjected to cryo-EM where mP-gp in the post-hydrolytic (ADP-bound, vanadate-trapped) state provided the highest resolution dataset that led to a reconstruction of 3D density map at the resolution of 7.9 Å which showed an inward- facing conformation. Rigid-body model fitting unveiled densities that were not accounted for by the fitted model illustrating new features such as bound ADP, extended NBD1- TMD2 linker and alternative allocrite-binding sites. Ultimately, the knowledge of P-gp conformation alteration was enhanced and a refined alternating access mechanism of P- gp was proposed based upon information derived from this study.

Estágio realizado na SONAE, e orientado pelo Eng. José Ferreira / Tese de Mestrado Integrado. Engenharia Industrial e Gestão. Faculdade de Engenharia. Universidade do Porto. 2012

Precast concrete bridge deck panels have been used for decades to accelerate bridge construction. Cracking of the transverse connection between panels is a common problem that can damage deck overlays and cause connection leaking leading to corrosion of lower bridge elements. To better understand the behavior of bridge deck transverse female-to-female connections, shear and moment lab testing were performed at Utah State University for the Utah Department of Transportation. Two existing UDOT connections were tested, a welded stud connection and a post tensioned connection. A variation of the welded connection using rebar was also tested. In addition, two new curved bolt connections were tested as a new method of post tensioning a connection. The manner of connection cracking and associated cracking loads were recorded along with the ultimate connection capacities. The connections were also tested in a low cycle, high amplitude cyclical shear test. Lab testing showed that the welded stud connection had the lowest moment capacity. It also showed that the welded rebar connection had significantly higher strength than the welded stud connection with higher cracking and ultimate loads. Curved bolts were also shown to be a good way to post tension a connection with similar moment capacities as the post tensioned connection. Longer curved bolts were found to perform better than shorter curved bolts.

Arzneistofftransporter ermöglichen endogenen und exogenen Molekülen die Überwindung von Zellmembranen und tragen dadurch zur Aufnahme, Verteilung und Elimination von Arzneistoffen bei. Inhalativ applizierte Wirkstoffe, wie Vertreter aus der Gruppe der Beta-2-Sympathomimetika oder Anticholinergika, zählen zu den Substraten wichtiger, pulmonal exprimierter Arzneistofftransporter. Trotz intensivierter Forschung auf dem Gebiet der Transporter-Expression ist diese im humanen Lungengewebe bisher wenig untersucht und deren pharmakokinetische Auswirkungen auf pulmonal verabreichte Arzneistoffe sind kaum bekannt. Im Rahmen der vorliegenden Arbeit sollte der Einfluss von Arzneistofftransportern auf die pulmonale Absorption inhalierter Arzneistoffe untersucht und Erkenntnisse über deren Expressions-Profil im humanen Lungengewebe gewonnen werden. Pharmakokinetische Parameter des inhalativen Anticholinergikums Ipratropiumbromid wurden an einem ex vivo Modell der humanen Lunge untersucht. Nach vorheriger Applikation des kompetitiven OCTN1/2-Inhibitors L-Carnitin wurde keine signifikante Reduktion der absorbierten Wirkstoffmenge detektiert. Damit zeigten sich die beiden organischen Kationen/Carnitin-Transporter OCTN1 und OCTN2, anders als bisher vermutet, nicht als primär an der Absorption von Ipratropiumbromid beteiligte Transporter. Infolgedessen wurde die Beteiligung weiterer Transporter hypothetisiert. Erstmals wurden die am humanen Lungen-Perfusions-Modell gewonnenen pharmakokinetischen Daten zur pulmonalen Absorption in direkter Beziehung zur mRNA- und Protein-Expression von Arzneistofftransportern in den jeweiligen individuellen Gewebeproben betrachtet. Die pulmonale Genexpression des Multidrug Resistance-Related Protein MRP5 wies eine signifikante negative Korrelation mit der Area under the curve (AUC0 – 60 min) von Ipratropiumbromid auf (r = -0,699; p < 0,05), was die Beteiligung von MRP5 an den Umverteilungsprozessen von Ipratropiumbromid in der humanen Lunge nahelegte. Auf Protein-Ebene wurde eine positive Korrelation zwischen der Expression des organischen Kationentransporters OCT3 und der AUC0 – 60 min von Ipratropiumbromid ermittelt (r = 0,7499,p < 0,05), woraus sich eine potentielle Beteiligung von OCT3 an der Aufnahme von Ipratropiumbromid aus dem luminalen Lungenbereich ableiten ließ. Zur Untermauerung dieser Hypothese wurden Untersuchungen mit stabil transfizierten HEK293-Zellen durchgeführt. Sowohl der organische Kationentransporter OCT1 als auch OCT3 trugen dabei signifikant zu einer erhöhten zellulären Aufnahme der beiden Tritium-markierten Bronchodilatatoren Ipratropiumbromid und Salbutamol bei. Damit wurde für OCT3 zum ersten Mal eine Beteiligung an der zellulären Aufnahme dieser beiden Arzneistoffe nachgewiesen. Im Kontext der Gendermedizin sind geschlechtsspezifische Unterschiede in der Transporter-Expression von großem Interesse. Inwiefern die drei Sexualsteroidhormone Estradiol, Progesteron und Testosteron einen regulatorischen Effekt auf die mRNA-Expression von Membrantransportern haben, wurde erstmals durch in vitro Inkubationsversuche in physiologischen Hormonkonzentrationen mit der humanen Bronchialepithelzelllinie Calu-3 geprüft. Mittels intensiv optimierter und sorgfältig validierter RT-qPCR-Analytik konnten vor allem nach Inkubation mit weiblichen Sexualhormonen verglichen zu keiner Hormon-Zugabe statistisch signifikante Expressions-Unterschiede detektiert werden: Nach Behandlung mit Estradiol zeigten der Oligopeptid-Transporter PEPT2 (80,8 ± 15,6 %) und OCTN2 (82,8 ± 4,2 %) eine geringere Genexpression, das Multidrug Resistance-Related Protein MRP1 (111,6 ± 9,1 %) sowie OCTN1 (112,9 ± 10,1 %) waren nach Zugabe von Estradiol kombiniert mit Progesteron höher exprimiert als ohne Hormon-Zusatz. Da Estradiol überdies als Inhibitor des OCT1- und OCT3-vermittelten Transports gilt, wurde die Auswirkung des Hormons, unter anderem in physiologischer Konzentration, auf die Aufnahme von Tritium-markierten Ipratropiumbromid in stabil transfizierte HEK293-Zellen untersucht, wobei tatsächlich eine reduzierte zelluläre Ipratropiumbromid-Aufnahme beobachtet wurde. Somit könnte auch in vivo eine geschlechtsspezifische Inhibition der beiden Transporter stattfinden, wodurch deren Substrate einer geschlechtsspezifisch variierenden Pharmakokinetik unterliegen könnten. Darüber hinaus wurde in rund 80 humanen Lungengewebsproben die Genexpression von Arzneistofftransportern hinsichtlich geschlechts- und altersspezifischer Unterschiede überprüft. In unter 50-jährigen Männern war das Multidrug-Resistance Protein MDR1 signifikant höher exprimiert verglichen zu Männern von 50 - 60 Jahren. OCT1 war in Patienten von 50 - 60 Jahren signifikant geringer exprimiert als in über 60-Jährigen. Daneben lieferte die Analyse aller Gewebeproben das Genexpressions-Profil von Arzneistofftransportern im humanen Lungengewebe, wobei OCT3 das höchste und OCT2 das geringste mRNA-Expressions-Niveau unter den untersuchten Transportern aufwies. Eine wesentliche Beteiligung von OCT3 an Transportvorgängen im humanen Lungengewebe erschien damit wahrscheinlich. Resümierend konnte mit der vorliegenden Arbeit ein Beitrag zur Aufklärung des Einflusses von Arzneistofftransportern auf die pulmonale Absorption inhalativ verabreichter Arzneistoffe geleistet werden. Dabei konnte OCT3 erstmals als maßgeblich an der zellulären Aufnahme von Ipratropiumbromid beteiligter Transporter in der humanen Lunge identifiziert werden, womit einerseits die Beteiligung von Arzneistofftransportern an pharmakokinetischen Prozessen in vivo und andererseits die Bedeutung von Arzneistofftransportern für die inhalative Arzneimitteltherapie deutlich wurde. / Drug transporters facilitate the transport of endogenous and exogenous compounds across cell membranes. Therefore they contribute to the absorption, distribution and elimination of drugs. Pulmonary administered drugs, such as members of the drug class of betamimetics or anticholinergics, are known substrates of relevant pulmonary expressed drug transporters. Despite intensified research in the field of transporter expression few data are available about their expression in the human lung and the pharmacokinetic implications on pulmonary administered drugs. The aim of this thesis was to investigate the impact of drug transporters on the absorption of inhaled drugs and to gain insights into their expression profiles in human lung tissue. Pharmacokinetic properties of the inhaled anticholinergic ipratropium bromide were explored using an ex vivo model of the human lung. After preceding application of the competitive OCTN1/2-inhibitor L-carnitine no significant decrease of the amount of absorbed active ingredient was detected. This contradicted previous assumptions regarding the contribution of the organic cation/carnitine transporters OCTN1 and OCTN2 to the absorption of ipratropium bromide. Consequently the involvement of additional transporters was hypothesized. For the first time pharmacokinetic data of the pulmonary absorption obtained by employing the human lung perfusion model were correlated with the mRNA and protein expression of drug transporters in respective individual tissue samples. The pulmonary gene expression of the multidrug resistance–related protein MRP5 showed a significant negative correlation with the area under the curve (AUC0 – 60 min) of ipratropium bromide (r = -0,699; p < 0,05). This might indicate that MRP5 contributes to the redistribution processes of ipratropium bromide in the human lung. A positive correlation between the protein expression of the organic cation transporter OCT3 and the AUC0 – 60 min of ipratropium bromide was detected (r = 0,7499, p < 0,05) suggesting a potential involvement of OCT3 in the absorption of ipratropium bromide in the luminal lung area. Uptake assays using stably transfected HEK293 cells were performed to substantiate this hypothesis. Both organic cation transporters, OCT1 and OCT3, contributed significantly to an increased cellular uptake of the tritium labeled bronchodilators ipratropium bromide and salbutamol. Thus, the contribution of OCT3 to the cellular uptake of both pharmaceutical substances was demonstrated for the first time. Gender-specific differences of drug transporter expression are of major interest in the context of gender medicine. In vitro incubation studies with the human bronchial epithelial cell line Calu-3 for the first time elucidated whether physiological concentrations of the three sex steroid hormones estradiol, progesterone and testosterone exert a regulatory effect upon the mRNA expression of membrane transporters. By thoroughly optimized and carefully validated RT-qPCR analytics statistically significant differences in gene expression were detected primarily after incubation with female sex hormones compared to no hormone exposure: After incubation with estradiol the peptide transporter PEPT2 (80,8 ± 15,6 %) and OCTN2 (82,8 ± 4,2 %) showed decreased expression whereas the multidrug resistance–related protein MRP1 (111,6 ± 9,1 %) as well as OCTN1 (112,9 ± 10,1 %) were upregulated after addition of both estradiol and progesterone compared to no treatment. Since estradiol is also a known inhibitor of the transport mediated by OCT1 and OCT3 its impact on the uptake of tritium labeled ipratropium bromide was investigated in stably transfected HEK293 cells. Indeed, a reduced cellular uptake of ipratropium bromide was observed after incubation with estradiol, also at physiological concentrations. Therefore, a gender-specific inhibition of both transporters in vivo is conceivable and could result in gender-specific pharmacokinetic characteristics for substrates of these transporters. Moreover, the gene expression of drug transporters in approximate 80 lung tissue samples was examined regarding gender and age related differences. The multidrug resistance protein MDR1 was significantly higher expressed in men younger than 50 years compared to 50 - 60 year old men. OCT1 was significantly less expressed in 50 - 60 years old patients compared to patients older than 60 years. Furthermore, the gene expression profile of drug transporters in the human lung was analyzed. In all tissue samples OCT3 showed the highest mRNA expression level whereas OCT2 was least expressed amongst the investigated transporters. This suggested a substantial involvement of OCT3 in transport processes in human lung tissue. In conclusion, the present research contributed to the elucidation of the role of drug transporters in the pulmonary absorption of inhaled drugs. For the first time OCT3 was identified to be substantially involved in the cellular absorption of ipratropium bromide in human lungs. Hence, the data supported the involvement of drug transporters in pharmacokinetic processes in vivo and emphasized the importance of drug transporters for inhaled pharmacotherapy.

Dans cette thèse, nous avons cherché à intégrer les perspectives de diffusion et d'adoption de l'innovation au sein d'un même modèle explicatif et nous avons appliqué ce modèle au cas de l'ABC en France. L'apport de notre recherche se voulait ainsi triple : contribution à l'étude de l'adoption de l'ABC, contribution à l'étude de la diffusion de l'ABC et intégration de ces deux perspectives au sein d'un même modèle explicatif.

<p>Bostadsaffären är för de allra flesta den största affären i livet. Varje år säljs ett stort antal fastigheter i Sverige. En stor del av dessa fastigheter säljs med hjälp av en traditionell fastighetsmäklare och de mäklarförmedlade fastigheterna säljs i de allra flesta fall med prissättningsmetoden provision. Provision är ett omtalat ämne som ofta möter motstånd. Syftet med vår uppsats var att ta reda på hur hem- och villaintresserade personer samt traditionella fastighetsmäklare ställer sig till provision. Syftet var även att ta reda på om provision verkligen är den ultimata prissättningsmetoden eller om det skulle kunna vara aktuellt att istället ta betalt per aktivitet. För att besvara våra frågor har vi vävt samman teori med vår empiri. Empirin har vi kommit fram till genom metodtriangulering. Slutsatsen av vår uppsats är att hem- och villaintresserade har en ganska negativ inställning till provision men att fastighetsmäklarna ser denna metod som den mest ultimata. Vi har kommit fram till att betalning per aktivitet inte skulle vara möjligt att använda med dagens arbetssätt men att fastighetsmäklarna snart måste förändra antingen hem- och villaintresserades bild av provision eller själva prissättningsmetoden provision.</p>

<p><strong>Syfte</strong>Att genom en fallstudie vid Gävle Energi belysa viktiga diskussioner i beslutsprocessen vid val av produktkalkyleringsmodell. Att ge Gävle Energis affärsområde Kommunikation ett förslag på modell för produktkalkylering, inklusive ett implementeringsförslag.</p><p><strong>Metod</strong>Vi har valt att genomföra en kvalitativ fallstudie som baseras på ett flertal intervjuer. Informationen som vi har samlat in under intervjuerna ligger till grund för avsnitten empiri, process och slutdiskussion. Vi har vidare studerat teorier för att få information till teorikapitlet samt till processen där empiri och teori förenas. I processen analyserar vi den information vi samlat in för att i slutet kunna redogöra vilka beslut som har varit avgörande vid valet av modell samt ge företaget ett förslag på lämplig modell.</p><p><strong>Resultat & slutsats</strong>Företaget efterfrågade en standardiserad enkel modell för produktkalkylering i en komplex verksamhet. Efter analysen rekommenderar vi totalstegskalkylering som modell och bifogar ett implementeringsförslag. Vad som genomgående har stött bort ABC‐kalkyleringen till fördel för totalstegskalkylering har varit företagets starka önskemål om enkelhet.</p>

The biological membrane surrounding the living cell provides a sealed barrier that tightly regulates the interactions with the outside environment. A large number of integral membrane proteins mediate these interactions and are involved in a wide variety of biological processes. An increasing number of studies have led to the conclusion that lipids provide more than a hydrophobic solvent for membrane proteins, and that interactions between lipids and proteins are required to allow protein function. ABC transporters are one of the most important family of membrane proteins. However, the importance of their lipidic environment is largely unknown. Only a few studies showed that their activity was dependent on the lipidic composition of the surrounding bilayer. The bacterial ABC transporter HorA was used as a model to probe the influence of the lipidic environment on that class of membrane proteins. HorA is a multidrug transporter expressed in Lactobacillus brevis, a Gram-positive beer spoilage bacterium. It turned out that phosphatidylethanolamine (PE) was indispensable to maintain both the activity and the structural integrity of HorA. Surprisingly, replacement of PE by the chemically related PC (phosphatidylcholine) did not led to the suppression of HorA activity, but to an unexpected phenotype. Whereas the cytoplasmic domains of HorA were still able to hydrolyze ATP, the membrane parts of the transporter were unable to use that energy to mediate substrate transport. Using several biophysical methods particularly adapted to the study of reconstituted systems, we showed that the structure of HorA is strongly altered by this lipid replacement. In particular, the structural organization of the transmembrane domains of the protein is strongly affected.

SyfteAtt genom en fallstudie vid Gävle Energi belysa viktiga diskussioner i beslutsprocessen vid val av produktkalkyleringsmodell. Att ge Gävle Energis affärsområde Kommunikation ett förslag på modell för produktkalkylering, inklusive ett implementeringsförslag. MetodVi har valt att genomföra en kvalitativ fallstudie som baseras på ett flertal intervjuer. Informationen som vi har samlat in under intervjuerna ligger till grund för avsnitten empiri, process och slutdiskussion. Vi har vidare studerat teorier för att få information till teorikapitlet samt till processen där empiri och teori förenas. I processen analyserar vi den information vi samlat in för att i slutet kunna redogöra vilka beslut som har varit avgörande vid valet av modell samt ge företaget ett förslag på lämplig modell. Resultat &amp; slutsatsFöretaget efterfrågade en standardiserad enkel modell för produktkalkylering i en komplex verksamhet. Efter analysen rekommenderar vi totalstegskalkylering som modell och bifogar ett implementeringsförslag. Vad som genomgående har stött bort ABC‐kalkyleringen till fördel för totalstegskalkylering har varit företagets starka önskemål om enkelhet.

Companies need different information about their operations, so that they can make better decisions and be more effective in the business world. One way to obtain that information is through looking at how costs are assigned to different cost objects, such as products or services. Of special interest is the allocation of indirect costs, because if these costs make up an important part of an organisation, then grasping them will help the firm be more competitive and respond better to customers and their needs. One method of allocating indirect costs is called activity based costing, or ABC, and it looks upon what activities that are being performed by a company, how much they cost based on resource usage, what drives the costs, and most importantly, it assigns these ex-penses to products/services. The aim of this thesis has been to investigate whether the ABC method can be applied in an event organising company. In order to reach the purpose we have used the example of Elmia AB, an event and trade show organiser situated in Jönköping, Sweden. The focus of our investigation was exhibition stands used in trade shows. We have come to the conclusion that activity based costing is applicable when it comes to event organisers in the sense that it is possible to identify major activities and depict re-sources. However, problems emerge when trying to estimate costs of resources because it is hard to know in advance what customers want, and therefore one cannot clearly distin-guish or set fixed and variable costs, nor can one easily cope with problems of unused ca-pacity, that is resources that are supplied but not used. This is especially the case with cus-tomised exhibition stands. The process is somewhat easier when it comes to standardised offerings, because they are less complicated since they are already set and cannot be much altered by the customers. Furthermore, cost drivers can be applied in this setting, at least theoretically because they provide help in determining what processes the customers might find important. On the other hand, they might be rather difficult to measure. Finally, the actual assigning of costs to cost objects is hard, because for the complex solutions, one cannot easily find common activities across different stands, but this can be easier to do when exhibition stand pack-ages are standardised and demand is better traceable. We have based our results on one company as a generalisation of event organisers as a whole, and we can argue that the example that we have chosen can be a good representa-tive of this particular branch of the service industry, because it shows how important indi-rect costs are to this dynamic business and also it reveals the importance of customers and their role when applying activity based costing to the environment. However, in order to have a complete conclusion with respect to our aim, more research is needed in other event and trade show organising firms because there are company specific situations in terms of size, structure, culture, etc, of a company that makes ABC a special case that changes ac-cording to different objectives of different users.